Richard,
We have purified and characterized these peptides already using conventional
techniques (HPLC, Edman degradation and MS). Now we have found that the peptides
are present in biological fluids from certain patients. We detect the peptides
using a dot blotting protocol and polyclonal antibody raised against the
peptides. However, we prefer to use the Tricine gels followed by immuno
blotting because we can then detect size variant very easily. The problem is
that the transfer efficiency is poor and I was thinking that maybe a low pH
blotting protocol could be used to make the peptides positively charged ? Are
you familiar with such a procedure ?
Thank you for your comments
jan
_______________________________________________________________________________
Subject: Re: Blotting of basic peptides
From: cook@MIT.EDU at internet
Date: 7/1/98 10:50
for one thing, don't use caps w/ basic proteins (use tris gly pH 7 buffer)
...caps buffer is ca. pH 11 and that may be close to the PI of your samples
so no movement will take place because there is no charge...this effect
occurs even though protein is coated w/ SDS..(in my observations) ...put
membranes on both sides of gel....these are very small peptides for
electrotransfer and that may be a factor too...use the "second generation
PVDF's like "proBlott and Biorad PVDF or PSEQ form Millipore that have
tighter pores and bind better don't use PVDF-P ....why not use HPLC to
isolate these peptides?
Good luck
Richard
>Dear ABRFers,
>
>
>We are trying to electrotransfer small (7-10 residues) basic peptides from
>Tricine-SDS gels to PVDF. We have tried the usual CAPS / 20% methanol
>procedure
>but the yield is low. Can anybody recommend a better blotting protocol for
>small
>basic peptides.
>
>Best Regards
>
>Jan J. Enghild
>
>
>----------------------------------------------------
>| |
>| Jan J. Enghild Ph.D |
>| Duke University Medical Center |
>| Department of Pathology P.O. Box 3712 |
>| Durham, NC, 27710 |
>| USA |
>| Phone: (919) 684 2872 |
>| Fax: (919) 684 2920 |
>| e-mail: enghi001@mc.duke.edu |
>| |
>| |
>----------------------------------------------------
***********************************
Richard F. Cook
HHMI CCR MIT Biopolymers Laboratory
Massachusetts Institute of Technology
40 Ames St.
E17-415
Cambridge, MA 02139
(617) 253-1685 Office
(617) 253-7038 Lab-1
(617) 253-1474 Lab-2
(617) 253-9398 FAX
talktome@biopolymers.mit.edu
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Subject: Re: Blotting of basic peptides
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